The present invention relates to a novel method of identifying Bt genes that are good candidates for producing a protein having insecticidal activity.
Bacillus thuringiensis (Bt) is a gram-positive soil bacterium characterized by its ability to produce parasporal crystal protein inclusions (delta endotoxins) during sporulation. These crystal inclusions contain proteins, many of which display a highly specific insecticidal activity. Most strains are active against caterpillar larvae of the order Lepidoptera. Several strains show activity against larvae of certain members of the order Diptera, and several strains have activity against some larvae of the order Coleoptera. Various Bt insecticidal proteins are reviewed in numerous prior art sources, including for example, Aronson et al., Microbiol. Rev. 50:1-24 (1986); Wilcox et al., Protein Engineering, Academic Press, New York, N.Y., pp. 395-413, (1986); Whiteley and Schnepf, Ann. Rev. Microbiol. 40:549-76 (1986), and Hofte and Whiteley, Microbiol. Rev. 53:242-255 (1989).
The prior art has disclosed various methods of identifying Bt strains producing insecticidal proteins. These methods include: 1) use of monoclonal antibodies, 2) insect bioassays, 3) biochemical assays, and 4) hybridization assays.
Monoclonal antibodies to insecticidal proteins of Bacillus thuringiensis have been made [Hofte et al., Applied Envir. Microbiol. 54:2010-2017 (1988)] and used to characterize various classes of insecticidal Bt proteins [Peferoen, Symposium on the Analytical Chemistry of Bacillus thuringiensis, American Chemical Society]. This technique has the disadvantage of requiring the lengthy identification, characterization and production of monoclonal antibodies. Monoclonal antibodies provide limited information about the genes producing the proteins that are recognized by a given antibody.
Insect bioassays have been used to characterize large numbers of Bacillus thuringiensis isolates. This technique has the disadvantage of being time consuming and labor intensive. In addition to limiting the number of strains characterized, this method does not readily discriminate among different genes and makes redundant testing of the same strains likely.
Biochemical assays have also been used to screen strains within the species Bacillus thuringiensis, [Martin et al., BioTechniques 3:386-392 (1985). These techniques have the disadvantage of not characterizing the genes encoding the insecticidal proteins of isolates and, therefore, not predicting insecticidal activities.
Hybridization assays have been used to characterize strains according to which of three major classes of Bt insecticidal genes they contain, [Kronstad and Whiteley, Gene 43:29-40 (1986)]. Hybridization techniques have the disadvantage of being labor intensive, time consuming, and are unable to discriminate different genes within the major insecticidal classes.
The polymerase chain reaction has been used to amplify genetic material and the details of the PCR technique are well known in the art. See, for example, Innis et al., PCR Protocols: A Guide to Methods and Applications, Academic Press, Inc., New York, 1990; and Erlich, PCR Technology Principles and Applications for DNA Amplification, Erlich, PCR Technology Principles and Applications for DNA Amplification, Stockton Press, New York, (1989), both of which are hereby incorporated by reference for their relevant teachings.
Wallace and Skolnick (WO 89/10414, published Nov. 2, 1989) discloses a method of using amplified sequence polymorphisms to detect variation in the genes amplified. Only human genes are exemplified and no correlation is made between the type of genetic variation and the effect on phenotypic trait.